Reverse genetics systems

ABSTRACT

The invention provides various reverse genetics systems for producing segmented RNA viruses, wherein the systems do not require bacteria for propagation of all of their expression constructs.

This patent application claims priority from U.S. provisional patentapplication 61/273,151, filed 31 Jul. 2009, the complete contents ofwhich are incorporated herein by reference.

TECHNICAL FIELD

This invention is in the field of reverse genetics. Furthermore, itrelates to preparing viruses e.g. for use in manufacturing vaccines forprotecting against various viruses.

BACKGROUND ART

Reverse genetics permits the recombinant expression and manipulation ofviruses in cell culture. It is a powerful tool in virology and vaccinemanufacture because it allows rapid production and/or mutation ofviruses, including reassortant production. The method involvestransfecting host cells with one or more plasmids which encode the viralgenome then isolating (or “rescuing”) virus from the cells. It can beused for the production of a wide variety of RNA viruses, includingpositive-strand RNA viruses [1,2], negative-strand RNA viruses [3,4] anddouble-stranded RNA viruses [5].

A drawback of known methods is that they rely on plasmids: Generatingthese plasmids requires cloning steps to be performed in bacteria, whichcan take several days or weeks to perform and verify for a segmented RNAvirus. Such delays interfere with the timetable for yearly production ofseasonal influenza vaccines and also prevent a rapid response to apandemic outbreak. Furthermore, the use of bacteria entails the riskthat bacterial contaminants might be introduced when the plasmids areused to transfect a host cell for virus production. These drawbacks areaddressed in reference 6 by using linear expression constructs insteadof plasmids. The linear expression constructs do not containamplification and/or selection sequences which are used during bacterialpropagation and almost always results in the molecular cloning of asingle representative of a viral quasispecies. Such linear expressionconstructs can be used to transfect host cells directly, giving a muchmore rapid reverse genetics system: reference 6 suggests thattransfection of the linear constructs can be achieved within hours ofreceiving a viral isolate, avoiding the time required for molecularcloning and allowing access to useful members of the original viralquasispecies population.

DISCLOSURE OF THE INVENTION

For a segmented virus the method used in reference 6 uses one linearconstruct per viral segment. Thus reverse genetics virus production bythis method requires transfection of a host cell with eight differentconstructs. An object of the invention is to avoid the need for suchmultiple transfections. More generally, it is an object of the inventionto provide further and improved methods for practising reverse geneticsfor segmented RNA viruses, and in particular to provide further methodswhich do not require the use of bacteria. The invention provides variousreverse genetics systems for producing segmented RNA viruses, whereinthe systems do not require bacteria for propagation of all of theirexpression constructs. Ideally, bacteria are not required at all.producing segmented RNA viruses, wherein the systems do not requirebacteria for propagation of all of their expression constructs. Ideally,bacteria are not required at all.

In a first aspect, a reverse genetics system is based on a non-bacterialexpression construct which encodes at least two viral genome segments.This system reduces the number of constructs which have to betransfected into a host cell for production of a complete viral genome.For instance, a single construct can be used to encode eight influenzavirus segments, thereby giving an 8-fold reduction in the complexity oftransfections as compared to reference 6. Thus the invention provides anon-bacterial expression construct comprising coding sequences forexpressing at least two different genome segments of a segmented RNAvirus. The invention also provides a eukaryotic host cell including thisnon-bacterial expression construct. The invention also provides a set oftwo or more such non-bacterial expression constructs, wherein the setencodes a complete segmented RNA virus genome.

In a second aspect, a reverse genetics system is based on a combinationof (i) at least one bacterial expression construct and (ii) at least onenon-bacterial expression construct. Each of these two types ofconstructs provides at least one viral genome segment. Although thisaspect does not totally avoid the use of bacteria for preparing thesystem, it is still powerful. For instance, constructs expressing asubset of the viral segments can be propagated and manipulated inbacteria, taking advantage of the wide range of convenient molecularbiological techniques which are available. The segments of this subsetcan be those which do not often need to be changed from strain tostrain. The remaining viral segments can be encoded by non-bacterialexpression constructs, and these constructs can be rapidly prepared atshort notice without requiring bacterial work. This combination thusmeans that efforts can focus on the segments of interest at shortnotice, and the constructs can be combined with an existing set of“background” segments which were already available. Thus the inventionprovides a set of expression constructs comprising (i) at least oneplasmid comprising coding sequence(s) for one or more genome segments ofa segmented RNA virus and (ii) at least one non-bacterial expressionconstruct comprising coding sequence(s) for one or more genome segmentsof the RNA virus, wherein the combination of bacterial and non-bacterialconstructs provides at least two different genome segments of the RNAvirus. The invention also provides a eukaryotic host cell including thisset of constructs.

In a third aspect, the invention provides a host cell including a linearexpression construct which comprises coding sequences for at least twodifferent genome segments of a segmented RNA virus. This cell may bebacterial but is preferably eukaryotic.

In a fourth aspect, the invention provides a bacterial plasmidcomprising coding sequences for eight different genome segments of aninfluenza virus, wherein expression of each segment is controlled byeither (i) a mammalian pol-I promoter or (ii) a bacteriophage polymerasepromoter. The invention also provides a cell including this construct,and this cell may be bacterial or eukaryotic.

The invention further provides a process for preparing a host cell ofthe invention, comprising a step of inserting into the cell one or moreexpression construct(s) mentioned above.

The invention further provides a process for RNA expression in aeukaryotic host cell of the invention, comprising a step of culturingthe host cell under conditions such that expression of the RNA virussegments occurs from the expression constructs.

The invention further provides a method for producing a segmented RNAvirus, comprising a step of culturing a host cell of the invention underconditions such that expression of the RNA virus segments occurs fromthe expression constructs to produce the virus. Virus produced in thisway may then be purified from the host cells or from a culture of thehost cells. The invention also provides virus obtained by this process.This virus may be used to infect eggs or cells to grow virus for vaccinemanufacture. Thus the invention provides a method for preparing a viralvaccine, comprising a step of infecting a culture host (e.g. eggs orcells) with a virus of the invention, growing the virus, and thenpreparing vaccine from the grown virus.

The invention also provides a process for preparing a DNA molecule whichcomprises coding sequences for expressing at least two differentsegments of a segmented RNA virus genome (e.g. a non-bacterialexpression construct of the invention), wherein the DNA is prepared atleast in part by chemical synthesis.

The invention also provides a process for preparing a DNA molecule whichcomprises coding sequences for expressing at least two differentsegments of a segmented RNA virus genome (e.g. a non-bacterialexpression construct of the invention), wherein the process comprisessteps of: (i) synthesising a plurality of overlapping fragments of theDNA molecule, wherein the overlapping fragments span the complete DNAmolecule; and (ii) joining the fragments to provide the DNA molecule.The DNA molecule may then be recovered and used in the reverse geneticsmethods of the invention e.g. it can be inserted into a eukaryotic cellfor generation of the segmented RNA virus. Preferably the DNA moleculeis not inserted into a bacterial cell between its recovery and itsinsertion into the eukaryotic cell i.e. the construct is used directlyfor viral rescue without any intermediate bacterial amplification.

The invention also provides a library of expression constructs for asegmented RNA virus, wherein each expression construct comprises acoding sequence for at least one genome segment of the virus. Thelibrary includes at least one construct for each segment of the genome,such that the whole genome can be represented by selecting a subset ofthe library. Some viral segments may be represented more frequently thanothers e.g. an influenza virus library may include many more HA and NAsegments than the average. To construct a desired viral genome ofinterest, library members encoding each desired segment are selected andthen expressed to give the desired virus. The library is particularlypowerful for influenza virus by permitting rapid reassortment ofbackbone genome segments with HA and NA segments of interest to producea useful virus for vaccine production.

Non-Bacterial Expression Constructs

The first, second and third aspects of the invention utilise one or more“non-bacterial expression constructs”. This term means that theconstruct can drive expression in a eukaryotic cell of viral RNAsegments encoded therein, but it does not include components which wouldbe required for propagation of the construct in bacteria. Thus theconstruct will not include a bacterial origin of replication (ori), andusually will not include a bacterial selection marker (e.g. anantibiotic resistance marker). These components are not required fordriving the desired viral RNA expression in a eukaryotic host cell andso are superfluous when bacteria are not used for propagation of theconstructs. Absence of these propagation components means that theconstructs will not be replicated if they are introduced into bacteria.

The non-bacterial construct may be linear or circular. Linear constructsare more usual (as seen in reference 6), but circular constructs canalso be used. Circular constructs can be made by circularising linearconstructs and vice versa. Methods for such circularisation aredescribed in ref. 6. Linearisation of a circular construct can beachieved in various easy ways e.g. utilising one or more restrictionenzyme(s), or by amplification from a template (including a circulartemplate) using a nucleic acid amplification technique (e.g. by PCR).

A non-bacterial construct includes coding sequence(s) for one or moreviral RNA segment(s). Constructs for the first and third aspects encodeat least two different viral RNA segments. The encoded segments can beexpressed and then function as viral RNAs which can be packaged intovirions to give recombinantly expressed virus. Thus the constructs aresuitable for producing a RNA virus by reverse genetics, either alone orin combination with other constructs.

The construct will usually be made of double-stranded DNA. Suchconstructs can conveniently be made by known methods of DNA synthesisand assembly. Modern techniques can provide synthetic DNA moleculesencoding a complete virus even if it has many genomic segments. Forexample, a construct expressing all eight segments of the influenzavirus genome requires about 25,000 base pairs (25 kbp) of DNA, which iswell within the capability of current construct synthesis e.g. reference7 reports chemical synthesis of a 32 kbp gene by assembly of individual˜5 kbp synthetic fragments, and reference 8 reports the production of a583 kbp synthetic chromosome via intermediate stages of about 5 kbp, 7kbp, 24 kbp, 72 kbp or 144 kbp long. See below for further details.

Such synthetic methods are the preferred way of providing constructs(and in particular of providing linear constructs). Instead of usingchemical synthesis, however, DNA for a construct can be prepared from aRNA virus by reverse transcription to provide a cDNA, and extra DNAsequences can then be joined to the cDNA (e.g. by ligation) or the cDNAcan be incorporated into a larger DNA construct. In some embodiments, amixture of enzymatic and chemical methods is used e.g. reversetranscription followed by chemical addition to the termini.

As well as being free from any bacterial propagation elements, thenon-bacterial construct may also be free from any bacterial DNAmodifications. Thus the construct may include no methylated adenineresidues, and any methylated cytosine residues will be in the context ofa CpG dinucleotide motif i.e. there will be no methylated cytosineswhich are not followed by a guanidine.

The construct can be introduced into a host cell by any suitabletransfection method e.g. by electroporation, lipofection, DEAE-dextran,calcium phosphate precipitation, liposomes, gene guns, microparticlebombardment or microinjection. Once transfected, the host cell willrecognise genetic elements in the construct and will begin to expressthe encoded viral RNA segments.

Construct Synthesis

As mentioned above, a DNA expression construct may be prepared bychemical synthesis at least in part. The construct comprises codingsequences for expressing at least two different segments of a segmentedRNA virus genome (and preferably for expressing the complete genome of asegmented RNA virus) and can conveniently be prepared using thesynthetic methods disclosed in reference 8.

The synthetic method may involve notionally splitting the desired DNAsequence into fragments. These fragments may again be notionally splitone or more times, eventually arriving at a set of fragments which areeach of a size which can be prepared by a chosen DNA synthesis methode.g. by phosphoramidite chemistry. These fragments are then synthesisedand joined to give the longer fragments from the notional splittingstage, and these longer fragments are then joined, etc. until thecomplete sequence is eventually prepared. In this way reference 8prepared a 583 kbp genome by assembling ˜10⁴ 50 mer oligonucleotides invarious stages. The 50 mers were assembled into cassettes 5-7 kb long,and these cassettes were then assembled into ˜24 kbp fragments, whichwere then assembled into ˜72 kbp fragments, then ˜144 kbp, then givingtwo ˜290 kbp constructs, which were finally joined to give the completegenome.

The fragments are designed to overlap, thereby permitting them toassemble in the correct order. For instance, the cassettes overlapped byat least 80 bp, thereby enabling their assembly into the ˜24 kbpfragments, etc. Thus the method involves the synthesis of a plurality ofoverlapping fragments of the desired DNA molecule, such that theoverlapping fragments span the complete DNA molecule. Both ends of eachfragment overlap with a neighbouring 5′ or 3′ fragment, except for theterminal fragments of a linear molecule where no overlap is required(but to synthesise a circular molecule, the two terminal fragmentsshould overlap). Fragments at each stage may be maintained as inserts invectors e.g. in plasmids or BAC or YAC vectors. Assembly of fragmentsduring the synthetic process can involve in vitro and/or in vivorecombination. For in vitro methods, digestion with a 3′ exonuclease canbe used to expose overhangs at the terminus of a fragment, andcomplementary overhangs in overlapping fragments can then be annealed,followed by joint repair (“chewback assembly”). For in vivo methods,overlapping clones can be assembled using e.g. the TAR cloning methoddisclosed in reference 8. For fragments <100 kbp (e.g. easily enough toencode all segments of an influenza virus genome) it is readily possibleto rely solely on in vitro recombination methods.

Other synthetic methods may be used. For instance, reference 7 disclosesa method in which fragments ˜5 kbp are synthesised and then assembledinto longer sequences by conventional cloning methods. Unpurified 40base synthetic oligonucleotides are built into 500-800-bp synthons byautomated PCR-based gene synthesis, and these synthons joined intomultisynthon ˜5 kbp segments using a small number of endonucleases and“ligation by selection.” These large segments can be subsequentlyassembled into longer sequences by conventional cloning. This method canreadily provide a 32 kbp DNA molecule, which is easily enough to encodea complete influenza virus.

Similarly, reference 9 discloses a method where a 32 kb molecule wasassembled from seven DNA fragments which spanned the complete sequence.The ends of the seven DNAs were engineered with unique junctions,thereby permitting assembly only of adjacent fragments. Theinterconnecting restriction site junctions at the ends of each DNA aresystematically removed assembly.

Once the complete DNA molecule has been assembled, it is purified andmay be inserted directly into eukaryotic cells for virus production,without involving an intermediate step where the DNA is present inside abacterium.

When prepared by these methods, a DNA expression construct of theinvention may include one or more “watermark” sequences. These aresequences which can be used to identify or encode information in theDNA. It can be in either noncoding or coding sequences. Most commonly,it encodes information within coding sequences without altering theamino acid sequences. For DNAs encoding segmented RNA viral genomes, anywatermark sequences are ideally included in intergenic sites becausesynonymous codon changes may have substantial biological effects forencoded RNA segments.

Plasmids

The second and fourth aspects of the invention involve the use ofplasmids. These plasmids can conveniently be propagated in bacteria andso include a bacterial origin of replication (ori) and usually alsoinclude a bacterial selection marker (e.g. an antibiotic resistancemarker). Thus the plasmids are readily distinguished (both by sequenceand by function) from the non-bacterial expression constructs discussedabove. In general terms, the plasmids may be the same as plasmidsalready known in the art for reverse genetics, but the prior art doesnot disclose their use in combination with non-bacterial expressionconstructs for virus rescue.

The plasmid also includes the necessary genetic elements to survive in aeukaryotic host cell, in which virus production can occur. Thus theplasmid is a shuttle plasmid which can be propagated, manipulated and/oramplified in a bacterial host but which can drive viral RNA expressionin a eukaryotic host.

The plasmid encodes at least one viral RNA segment (eight influenzavirus segments in the fourth aspect) and in a eukaryotic host cell thesecoding sequences can be expressed and then function as viral RNAs whichcan be packaged into virions to give recombinantly expressed virus.

The plasmid can be introduced into a eukaryotic host cell by anysuitable transfection method e.g. by electroporation, lipofection, geneguns, or microinjection. Once transfected, the host cell will recognisegenetic elements in the construct and will begin to express the encodedviral RNA segment(s).

When a plasmid encodes multiple RNA segments, steps can be taken tominimise intraplasmid recombination. The presence of multiple identicalpromoters and terminators (both pol-I and pol-II) can increase thisrisk, as can the use of a ori which provides a high copy number duringbacterial propagation. Thus a plasmid may advantageously have arelatively low copy number when present in bacteria e.g. <50 copies perE. coli cell. Various low copy number vectors are available e.g. thevectors used in reference 10, vectors which include the p15a ori or aplasmid F ori [11], etc. It is also useful to use promoters havingdifferent sequences and/or to avoid including superfluous coding regionswhich provide extra promoters. Steps such as these can improve thestability of a plasmid.

Expression Constructs

Non-bacterial and plasmid expression constructs used with the inventionencode viral RNA segment(s). These coding sequences can be expressed ina suitable eukaryotic host cell to provide viral RNAs which can bepackaged into virions to give recombinantly expressed virus.

Expression of a viral RNA segment will be controlled by a promoterupstream of the RNA-encoding sequence. The promoter for expressing aviral RNA segment in an animal cell will be recognised by aDNA-dependent RNA polymerase and will usually be a pol-I promoter (seebelow). Other systems are available, however, and it is known to usebacteriophage or bacterial RNA polymerase promoters, such as the T7 RNApolymerase, in association with an in situ source of the polymerase[12]. Each viral segment has its own promoter, and these may be the sameor different as each other.

Where the virus is a positive-strand RNA virus it is often sufficient totransfect a cell with an expression construct encoding only the viralsegments. For example, the transfection of plasmids encoding thepoliovirus genome resulted in the recovery of infectious poliovirus[1,2]. Reverse genetics for negative-strand RNA viruses presents extrachallenges because the antisense viral RNA is usually non-infective andthus requires viral proteins to complete the life cycle. Thus viralproteins such as the viral polymerase are supplied to the cell, eitherdelivered as protein or as a gene for in situ protein expression.

Thus an expression construct may include coding sequences for expressingviral proteins in eukaryotic cells, particularly for negative-strandviruses. Suitable promoters for protein expression include those fromcytomegalovirus (CMV). Co-expression of the viral segments and viralproteins gives all of the necessary elements in situ for recombinantassembly of a virus in the host cell. It is useful to include theprotein-coding sequences on the same construct as the RNA-codingsequences, but it is also possible to use different constructs for RNAand protein expression. Where the protein-coding and RNA-codingsequences are in the same construct, they may be different sequences butit is instead possible to drive expression from two different promotersto provide both RNA and protein expression from the same DNA sequence.

Bi-directional constructs are known in the art for expressing viral RNAfrom a pol-I promoter and viral protein from a pol-II promoter attachedto the same DNA sequence (e.g. see reference 13). The two promotersdrive expression in different directions (i.e. both 5′ to 3′ and 3′ to5′) from the same construct and can be on different strands of the samedouble stranded DNA. The use of a common DNA sequence reduces the totalnumber and/or length of expression constructs required by the host cell.A bi-directional expression construct can include a gene or cDNA locatedbetween an upstream pol-II promoter and a downstream pol-I promoter.Transcription of the gene or cDNA from the pol-II promoter producescapped positive-sense viral mRNA which can be translated into a viralprotein, while transcription from the pol-I promoter produces uncappednegative-sense vRNA.

An expression construct will typically include a RNA transcriptiontermination sequence for each transcription unit. The terminationsequence may be an endogenous termination sequence or a terminationsequence which is not endogenous to the host cell. Suitable terminationsequences will be evident to those of skill in the art and include, butare not limited to, RNA polymerase I transcription terminationsequences, RNA polymerase II transcription termination sequences, andribozymes. Furthermore, the expression constructs may contain one ormore polyadenylation signals for mRNAs, particularly at the end of agene used for protein expression. The coding sequences for viral RNAsegments are typically flanked by a pol-I promoter at one end and apol-II promoter at the other end, with pol-I promoter and terminatorsequences flanking the segment-encoding sequence, flanked in turn bypol-II promoter and terminator sequences. The spacing of these varioussequence elements with reference to each other is important for thepolymerase to correctly initiate and terminate replication, but this isnot difficult to achieve.

An expression construct may include a selectable marker for selection ineukaryotic cells.

An expression construct may include one or more multiple cloning sitesto facilitate introduction of a DNA sequence.

Where separate coding sequences are used for viral RNAs and proteins, itis possible to use different sequences e.g. the protein-coding sequencecould be codon-optimised for a particular host cell, whereas theRNA-coding sequence uses the codons natural to the virus in question.Codon optimisation of a RNA-coding sequence is less useful because theRNA should be optimal for virion packaging rather than for recombinantprotein expression.

Where the expression host is a canine cell, such as a MDCK cell line,protein-coding regions may be optimised for canine expression e.g. usinga pol-II promoter from a wild-type canine gene or from a canine virus,and/or having codon usage more suitable for canine cells than for humancells. For instance, whereas human genes slightly favour UUC as thecodon for Phe (54%), in canine cells the preference is stronger (59%).Similarly, whereas there is no majority preference for Ile codons inhuman cells, 53% of canine codons use AUC for Ile. Canine viruses, suchas canine parvovirus (a ssDNA virus) can also provide guidance for codonoptimisation e.g. 95% of Phe codons in canine parvovirus sequences areUUU (vs. 41% in the canine genome), 68% of Ile codons are AUU (vs. 32%),46% of Val codons are GUU (vs. 14%), 72% of Pro codons are CCA (vs.25%), 87% of Tyr codons are UAU (vs. 40%), 87% of His codons are CAU(vs. 39%), 92% of Gln codons are CAA (vs. 25%), 81% of Glu codons areGAA (vs. 40%), 94% of Cys codons are UGU (vs. 42%), only 1% of Sercodons are UCU (vs. 24%), CCC is never used for Phe and UAG is neverused as a stop codon. Thus protein-coding genes can be made more likegenes which nature has already optimised for expression in canine cells,thereby facilitating expression.

RNA Polymerase I Promoters

Most reverse genetics methods use expression vectors which comprise aRNA polymerase I (RNA pol-I) promoter to drive transcription of viralRNA segments. The pol-I promoter gives a transcript with unmodified 5′and 3′ ends which is necessary for full infectivity of many viruses e.g.influenza.

Natural pol-I promoters are bipartite, having two separate regions: thecore promoter and the upstream promoter element (UPE). Although thisgeneral organisation is common to pol-I promoters from most species,however, the actual sequences of the promoters vary widely. The corepromoter surrounds the transcription startpoint, extending from about−45 to +20, and is sufficient to initiate transcription. The corepromoter is generally GC rich. Although the core promoter alone issufficient to initiate transcription, the promoter's efficiency is verymuch increased by the UPE. The UPE typically extends from about −180 to−107 and is also GC rich. The activity of the promoter may be furtherenhanced by the presence of distal enhancer-like sequences, which mightfunction by stabilizing the pre-initiation complex.

The sequences of pol-I promoters have been identified in a variety ofspecies, including human, dog and chicken. The invention will typicallyuse a pol-I promoter which is endogenous to the host cell, as theactivity of pol-I promoters can be restricted to a narrow host range. Insome circumstances, however, a pol-I promoter can be active outside itsnatural host e.g. human pol-I promoters can be active in monkey cells,and also in some dog cells.

Expression constructs can include at least one core promoter; preferablythey also include at least one UPE, and they may also include one ormore enhancer elements. It is also possible to use the fragments ofnatural promoters, provided that these fragments can initiatetranscription. A human pol-I promoter which can be used according to theinvention may comprise the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, ora variant thereof. Where a canine promoter is used according to theinvention, it may comprise the sequence of SEQ ID NO: 3, SEQ ID NO: 4 orSEQ ID NO: 5, or a variant thereof. Canine pol-I promoters for reversegenetics are disclosed in references 14 & 15.

The pol-I promoter may comprise (i) a sequence having at least p %sequence identity to any of SEQ ID NOs: 1 to 5, and/or (ii) a fragmentany of SEQ ID NOs: 1 to 5, provided that the promoter has the ability toinitiate and drive transcription of an operatively linked RNA-encodingsequence in a host cell of interest. The value of p may be 75, 80, 85,90, 95, 96, 97, 98, 99 or more. The fragment may itself be of sufficientlength to drive expression (e.g. SEQ ID NO: 4 is a fragment of SEQ IDNO: 3) or the fragment may be joined to other sequences and thiscombination will drive expression. The ability of such pol-I promotersto drive expression in a host cell of interest can readily be assessede.g. using the assays described above with an antisense reporter geneunder control of the promoter.

Virus Preparation

The invention is useful for the production of virus strains, includingmodified or reassortant strains. The technique can use in vitromanipulation of DNA constructs to generate combinations of viralsegments, to facilitate manipulation of coding or non-coding sequencesin the viral segments, to introduce mutations, etc. The production ofreassortant virus strains is useful as it can significantly decrease thetime needed to obtain a reassortant seed virus which is particularlybeneficial in situations where a rapid production of vaccine is neededto counteract an epidemic. Thus, it is preferred that expressionconstructs are used to express viral segments from or derived from atleast two different wild-type strains.

In order to produce a recombinant virus, a cell must express allsegments of the viral genome which are necessary to assemble a virion.DNA cloned into the expression constructs of the invention preferablyprovides all of the viral RNA and proteins, but it is also possible touse a helper virus to provide some of the RNA and proteins, althoughsystems which do not use a helper virus are preferred.

To provide all viral segments from the constructs of the invention,various arrangements are possible. According to the first aspect, allviral segments can be encoded on non-bacterial expression constructs,provided that at least one of these constructs encodes at least twoviral genome segments (unlike reference 6); and ideally, all viralgenome segments are encoded on a single non-bacterial construct, suchthat transfection with that single construct is enough to provide a hostcell with the ability to produce the virus of interest. In contrast,according to the second aspect the viral segments are split betweenbacterial and non-bacterial expression constructs, and their combinedpresence in a cell provides expression of all viral segments.

It can be advantageous to split the viral segments between more than oneexpression construct, even with the first aspect. Taking vaccineproduction strains of influenza A virus as an example, six of the eightsegments typically do not change from year to year, and every seasonthis constant viral backbone is supplemented by seasonal HA and NAsegments. In this situation it can be helpful to encode the six backbonesegments on one construct, and to encode the two other variable segmentseither together on a second construct or separately on a second andthird construct. This permits the seasonal variations to be performed ona smaller construct, and also allows the backbone construct to beoptimised specifically for backbone expression.

Viruses

The methods of the invention may be practised with any segmented RNAvirus. Such viruses can be positive-stranded, negative-stranded, ordouble-stranded.

Where the virus is a negative-strand RNA virus, the virus may be from afamily selected from the group consisting of Paramyxoviridae,Pneumovirinae, Rhabdoviridae, Filoviridae, Bornaviridae,Orthomyxoviridae, Bunyaviridae, or Arenaviridae. Furthermore, the virusmay be a virus from a genus selected from the group consisting ofParamyxovirus, Orthomyxovirus, Respirovirus, Morbillivirus, Rubulavirus,Henipaviras, Avulavirus, Pneumovirus, Metapneumovirus, Vesiculovirus,Lyssavirus, Ephemerovirus, Cytorhabdovirus, Nucleorhabdovirus,Novirhabdovirus, Marburgvirus, Ebolavirus, Bomavirus, Influenzavirus A,Influenzavirus B, Influenzavirus C, Thogotovirus, Isavirus,Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, Tospovirus,Arenavirus, Ophiovirus, Tenuivirus, or Deltavirus. In specificembodiments, the negative-strand RNA virus is selected from the groupconsisting of Sendai virus, Measles virus, Mumps virus, Hendra virus,Newcastle disease virus, Human respiratory syncytial virus, Avianpneumovirus, Vesicular stomatitis Indiana virus, Rabies virus, Bovineephemeral fever virus, Lettuce necrotic yellows virus, Potato yellowdwarf virus, Infectious hematopoietic necrosis virus, Lake Victoriamarburgvirus, Zaire ebolavirus, Boma disease virus, Influenza virus,Thogoto virus, Infectious salmon anemia virus, Bunyamwera virus, Hantaanvirus, Dugbe virus, Rift Valley fever virus, Tomato spotted wilt virus,Lymphocytic choriomeningitis virus, Citrus psorosis virus, Rice stripevirus, and Hepatitis delta virus. In preferred embodiments, the virus isan influenza virus (see below).

Where the virus is a positive-strand RNA virus, the virus may be from afamily selected from the group consisting of Arteriviridae,Coronaviridae, Picornaviridae and Roniviridae. Furthermore, the virusmay be a virus from a genus selected from the group consisting ofArterivirius, Coronavirus, Enterovirus, Torovirus, Okavirus, Rhinovirus,Hepatovirus, Cardiovirus, Aphthovirus, Parechovirus, Erbovirus,Kobuvirus and Teschovirus. In specific embodiments, the virus isselected from the group consisting of severe acute respiratory syndrome(SARS) virus, polio virus, Human enterovirus A (HEV-A), Humanenterovirus B (HEV-B), Human enterovirus C, Human enterovirus D,Hepatitis A and Human rhinovirus A and B.

Where the virus is a double-stranded RNA virus, the virus may be from afamily selected from the group consisting of Birnaviridae, Cystoviridae,Hypoviridae, Partitiviridae, Reoviridae and Totiviridae. Furthermore,the virus may be a virus from a genus selected from the group consistingof Aquabirnavirus, Avibimavirus, Entomobirnavirus, Cystovirus,Partitivirus, Alphacryptovirus, Betacryptovirus, Aquareovirus,Coltivirus, Cypovirus, Fijivirus, Idnoreovirus, Mycoreovirus, Orbivirus,Orthoreovirus, Oryzavirus, Phytoreovirus, Rotavirus and Seadornavirus.

The present invention is particularly suitable for viruses which undergorapid mutation and where the recombinant approach allows for a morerapid isolation of the virus which can then be further propagated toobtain suitable vaccines. Therefore, in a preferred embodiment the virusis influenza.

Influenza Viruses

The invention is particularly suitable for use with influenza A virusand influenza B virus, for which reverse genetics has been wellcharacterized. Influenza viruses are segmented negative strand RNAviruses. Influenza A and B viruses have eight segments (PB2, PB1, PA,HA, NP, NA, M and NS), whereas influenza C virus has seven (no NAsegment). The virus usually requires the presence of at least four viralproteins (PB1, PB2, PA and nucleoprotein) to initiate replication. Atleast these four viral proteins should thus be provided byprotein-encoding expression constructs.

Preferred expression systems for influenza A viruses encode genomesegments derived from a plurality of different wild-type strains. Thesystem may encode 1 or more (e.g. 1, 2, 3, 4, 5 or 6) genome segmentsfrom a PR/8/34 strain (A/Puerto Rico/8/34), but usually this/these willnot include the PR/8/34 HA segment and usually will not include thePR/8/34 NA segment. Thus the system may encode at least one of segmentsNP, M, NS, PA, PB1 and/or PB2 (possibly all six) from PR/8/34.

Other useful expression systems for influenza A viruses may encode 1 ormore (e.g. 1, 2, 3, 4, 5 or 6) genome segments from an AA/6/60 influenzavirus (A/Ann Arbor/6/60), but usually this/these will not include theAA/6/60 HA segment and usually will not include the AA/6/60 NA segment.Thus the system may encode at least one of segments NP, M, NS, PA, PB1and/or PB2 (possibly all six) from AA/6/60.

Expression systems for influenza B viruses may encode genome segmentsderived from a plurality of different wild-type strains. The system mayencode 1 or more (e.g. 1, 2, 3, 4, 5 or 6) genome segments from aAA/1/66 influenza virus (B/Ann Arbor/1/66), but usually this/these willnot include the AA/1/66 HA segment and usually will not include theAA/1/66 NA segment. Thus the system may encode at least one of segmentsNP, M, NS, PA, PB1 and/or PB2 from AA/1/66.

Viral segments and sequences from the A/PR/8/34, A/AA/6/60, andB/AA/1/66 strains are widely available. Their sequences are available onthe public databases e.g. GI:89779337, GI:89779334, GI:89779332,GI:89779320, GI:89779327, GI:89779325, GI:89779322, GI:89779329.

In some embodiments it may be advantageous to provide an influenza viruswhose genome does not encode a NS1 viral protein, or whose NS1 proteinis truncated. NS1 knockout mutants are described in reference 16.Truncations are known in the art (e.g. see references 17 & 18) andinclude truncations which leave only the first N-terminal 126 aminoacids of NS1. These NS1-mutant virus strains are particularly suitablefor preparing live attenuated influenza vaccines.

A reverse genetics system for influenza virus (and certain otherviruses) may include an expression construct which leads to expressionof an accessory protein in the host cell. For instance, it can beadvantageous to express a non-viral serine protease (e.g. trypsin).

As mentioned above, it can be advantageous to split viral segmentsbetween several expression constructs. This is also true for influenzavirus.

In one embodiment, a first non-bacterial expression construct comprisescoding sequences for influenza virus A or B genome segments PB2, PB1,PA, NP and NS. A second non-bacterial construct comprises a codingsequence for influenza virus A or B genome segment HA. The NA and Mgenome segments are encoded either on the first construct (to give a“7:1” system) or on the second construct (to give a 5:3 system), or theM segment is on the first construct and the NA segment is on the secondconstruct (6:2). For influenza A virus, the first construct ideallyencodes segments from a PR/8/34, AA/6/60 or AA/1/66 strain. The segmentsencoded on the second construct can come from different strain(s) fromthe segments on the first construct, thereby facilitating the strainreassortment which is regularly performed prior to influenza vaccinemanufacture. Each of the coding sequences for the eight viral segmentshas a promoter for driving its expression as a vRNA e.g. a pol-Ipromoter. The first construct should also comprise coding sequences forexpressing at least the PB1, PB2, PA and NP viral proteins e.g. eachunder the control of a pol-II promoter. Usefully, to reduce the overalllength of the construct (thus increasing stability), the codingsequences for at least the PB1, PB2, PA and NP segments are flanked by apol-I promoter at one end and a pol-II promoter at the other end, suchthat bidirectional expression can provide the viral segments and theviral proteins from the same DNA coding sequence. Thus pol-I promoterand terminator sequences may flank the sequence encoding the viralsegment, and these may be surrounded by pol-II promoter and terminatorsequences. The pair of linear constructs can be transfected into animalcells which recognise the pol-I and pol-II promoters (e.g. intomammalian cells such as MDCK or PER.C6 cells) to provide infectiousinfluenza virus.

In another embodiment, a bacterial plasmid comprises coding sequencesfor influenza virus A or B genome segments PB2, PB1, PA, NP and NS. Anon-bacterial construct (preferably linear) comprises a coding sequencefor influenza virus A or B genome segment HA. The NA and M genomesegments are encoded either on the plasmid (to give a “7:1” system) oron the non-bacterial construct (to give a 5:3 system), or the M segmentis on the plasmid and the NA segment is on the non-bacterial construct(6:2). For influenza A virus, the plasmid ideally encodes segments froma PR/8/34, AA/6/60 or AA/I/66 strain. The segments encoded on thenon-bacterial construct can come from different strain(s) from thesegments on the plasmid, thereby facilitating the strain reassortmentwhich is regularly performed prior to influenza vaccine manufacture.Each of the coding sequences for the eight viral segments has a promoterfor driving its expression as a vRNA e.g. a pol-I promoter. The plasmidshould also comprise coding sequences for expressing at least the PB1,PB2, PA and NP viral proteins e.g. each under the control of a pol-IIpromoter. Usefully, to reduce the overall length of the plasmid, thecoding sequences for at least the PB1, PB2, PA and NP segments areflanked by a pol-I promoter at one end and a pol-II promoter at theother end, such that bidirectional expression can provide the viralsegments and the viral proteins from the same DNA coding sequence. Thuspol-I promoter and terminator sequences may flank the sequence encodingthe viral segment, and these may be surrounded by pol-II promoter andterminator sequences. The plasmid and the non-bacterial constructs aremaintained separately prior to use, but can then both be transfectedinto animal cells which recognise the pol-I and pol-II promoters (e.g.into mammalian cells such as MDCK or PER.C6 cells) to provide infectiousinfluenza virus.

In another embodiment, a non-bacterial construct (preferably linear)comprises coding sequences for influenza virus A or B genome segmentsPB2, PB1, PA, NP and NS. A bacterial plasmid comprises a coding sequencefor influenza virus A or B genome segment HA. The NA and M genomesegments are encoded: on the non-bacterial construct (to give a “7:1”system); or on the plasmid (to give a 5:3 system); or on separateplasmids (to give a 5:1:1:1 system); or the NA segment is on the sameplasmid as HA while the M segment is on the non-bacterial construct(6:2); or the NA segment is on a second plasmid and the M segment is onthe non-bacterial construct (6:1:1). For influenza A virus, thenon-bacterial construct ideally encodes segments from a PR/8/34, AA/6/60or AA/1/66 strain. The segments encoded on the plasmid can come fromdifferent strain(s) from the segments on the non-bacterial construct,thereby facilitating the strain reassortment which is regularlyperformed prior to influenza vaccine manufacture. Each of the codingsequences for the eight viral segments has a promoter for driving itsexpression as a vRNA e.g. a pol-I promoter. The non-bacterial constructshould also comprise coding sequences for expressing at least the PB1,PB2, PA and NP viral proteins e.g. each under the control of a pol-IIpromoter. Usefully, to reduce the overall length of the non-bacterialconstruct, the coding sequences for at least the PB1, PB2, PA and NPsegments are flanked by a pol-I promoter at one end and a pol-IIpromoter at the other end, such that bidirectional expression canprovide the viral segments and the viral proteins from the same DNAcoding sequence. Thus pol-I promoter and terminator sequences may flankthe sequence encoding the viral segment, and these may be surrounded bypol-II promoter and terminator sequences. The plasmid and thenon-bacterial constructs are maintained separately prior to use, but canthen both be transfected into animal cells which recognise the pol-I andpol-II promoters (e.g. into mammalian cells such as MDCK or PER.C6cells) to provide infectious influenza virus.

In some embodiments, however, a single construct is used to encode thecomplete viral genome. Thus the invention provides a non-bacterialexpression construct comprising coding sequences for expressing alleight influenza virus A or B genome segments. This construct is ideallya linear construct e.g. between 22-26 kbp. Each of the coding sequencesfor the eight viral segments has a promoter for driving its expressionas a vRNA e.g. a pol-I promoter. The construct should also comprisecoding sequences for expressing at least the PB1, PB2, PA and NP viralproteins e.g. each under the control of a pol-II promoter. Usefully, toreduce the overall length of the construct, the coding sequences for thePB1, PB2, PA and NP segments (and preferably for all eight viralsegments) are flanked by a pol-I promoter at one end and a pol-IIpromoter at the other end, such that bidirectional expression canprovide the viral segments and the viral proteins from the same DNAcoding sequence. Thus pol-I promoter and terminator sequences may flankthe sequence encoding the viral segment, and these may be surrounded bypol-II promoter and terminator sequences. This linear construct can betransfected into animal cells which recognise the pol-I and pol-IIpromoters (e.g. into mammalian cells such as MDCK or PER.C6 cells) toprovide infectious influenza virus.

Cells

The present invention can be performed in any cell that can express thevirus of interest after transfection with the expression construct(s).The invention will typically use a cell line, although primary cells maybe used as an alternative. The cell will typically be mammalian,although avian or insect cells can also be used. Suitable mammaliancells include, but are not limited to, hamster, cattle, primate(including humans and monkeys) and dog cells. Various cell types may beused, such as kidney cells, fibroblasts, retinal cells, lung cells, etc.Examples of suitable hamster cells are the cell lines having the namesBHK21 or HKCC. Suitable monkey cells are e.g. African green monkeycells, such as kidney cells as in the Vero cell line [19-21]. Suitabledog cells are e.g. kidney cells, as in the CLDK and MDCK cell lines.Suitable avian cells include the EBx cell line derived from chickenembryonic stem cells, EB45, EB14, and EB14-074 [22].

Further suitable cells include, but are not limited to: CHO; 293T; MRC5; PER.C6 [23]; FRhL2; W1-38; etc. Suitable cells are widely availablee.g. from the American Type Cell Culture (ATCC) collection [24], fromthe Coriell Cell Repositories [25], or from the European Collection ofCell Cultures (ECACC). For example, the ATCC supplies various differentVero cells under catalogue numbers CCL 81, CCL 81.2, CRL 1586 andCRL-1587, and it supplies MDCK cells under catalogue number CCL 34.PER.C6 is available from the ECACC under deposit number 96022940.

Preferred cells (particularly for growing influenza viruses) for use inthe invention are MDCK cells [26-28], derived from Madin Darby caninekidney. The original MDCK cells are available from the ATCC as CCL 34.It is preferred that derivatives of these or other MDCK cells are used.Such derivatives were described, for instance, in reference 26 whichdiscloses MDCK cells that were adapted for growth in suspension culture(‘MDCK 33016’ or ‘33016-PF’, deposited as DSM ACC 2219). Furthermore,reference 29 discloses MDCK-derived cells that grow in suspension inserum free culture (‘B-702’, deposited as FERM BP-7449). In someembodiments, the MDCK cell line used may be tumorigenic, but it is alsoenvisioned to use non-tumorigenic MDCK cells. For example, reference 30discloses non-tumorigenic MDCK cells, including ‘MDCK-S’ (ATCCPTA-6500), ‘MDCK-SF101’ (ATCC PTA-6501), ‘MDCK-SF102’ (ATCC PTA-6502)and ‘MDCK-SF103’ (ATCC PTA-6503). Reference 31 discloses MDCK cells withhigh susceptibility to infection, including ‘MDCK.5F1’ cells (ATCC CRL12042).

It is possible to use a mixture of more than one cell type for viralrescue, but it is preferred to use a single cell type e.g. usingmonoclonal cells. Preferably, the cells are from a single cell line. Thesame cell line may be used downstream for subsequent propagation of thevirus e.g. during virus growth.

Preferably, the cells are cultured in the absence of serum, to avoid acommon source of contaminants. Various serum-free media for eukaryoticcell culture are known to the person skilled in the art e.g. Iscove'smedium, ultra CHO medium (BioWhittaker), EX-CELL (JRH Biosciences).Furthermore, protein-free media may be used e.g. PF-CHO (JRHBiosciences). Otherwise, the cells for replication can also be culturedin the customary serum-containing media (e.g. MEM or DMEM medium with0.5% to 10% of fetal calf serum).

The cells may be in adherent culture or in suspension.

Vaccines

The invention provides an influenza virus produced by a host cell of theinvention. This influenza virus may be used in various ways e.g. as aseed virus for vaccine manufacture.

Thus the invention can utilise a rescued virus to produce vaccines.

Vaccines (particularly for influenza virus) are generally based eitheron live virus or on inactivated virus e.g. see chapters 17 & 18 ofreference 32. Inactivated vaccines may be based on whole virions,‘split’ virions, or on purified surface antigens. Antigens can also bepresented in the form of virosomes. The invention can be used formanufacturing any of these types of vaccine.

Where an inactivated virus is used, the vaccine may comprise wholevirion, split virion, or purified surface antigens (for influenza,including hemagglutinin and, usually, also including neuraminidase).Chemical means for inactivating a virus include treatment with aneffective amount of one or more of the following agents: detergents,formaldehyde, β-propiolactone, methylene blue, psoralen,carboxyfullerene (C60), binary ethylamine, acetyl ethyleneimine, orcombinations thereof. Non-chemical methods of viral inactivation areknown in the art, such as for example UV light or gamma irradiation.

Virions can be harvested from virus-containing fluids, e.g. allantoicfluid or cell culture supernatant, by various methods. For example, apurification process may involve zonal centrifugation using a linearsucrose gradient solution that includes detergent to disrupt thevirions. Antigens may then be purified, after optional dilution, bydiafiltration.

Split virions are obtained by treating purified virions with detergents(e.g. ethyl ether, polysorbate 80, deoxycholate, tri-N-butyl phosphate,Triton X-100, Triton N101, cetyltrimethylammonium bromide, Tergitol NP9,etc.) to produce subvirion preparations, including the ‘Tween-ether’splitting process. Methods of splitting influenza viruses, for exampleare well known in the art e.g. see refs. 33-38, etc. Splitting of thevirus is typically carried out by disrupting or fragmenting whole virus,whether infectious or non-infectious with a disrupting concentration ofa splitting agent. The disruption results in a full or partialsolubilisation of the virus proteins, altering the integrity of thevirus. Preferred splitting agents are non-ionic and ionic (e.g.cationic) surfactants e.g. alkylglycosides, alkylthioglycosides, acylsugars, sulphobetaines, betains, polyoxyethylenealkylethers,N,N-dialkyl-Glucamides, Hecameg, alkylphenoxy-polyethoxyethanols, NP9,quaternary ammonium compounds, sarcosyl, CTABs (cetyl trimethyl ammoniumbromides), tri-N-butyl phosphate, Cetavlon, myristyltrimethylammoniumsalts, lipofectin, lipofectamine, and DOT-MA, the octyl- or nonylphenoxypolyoxyethanols (e.g. the Triton surfactants, such as Triton X-100 orTriton N101), polyoxyethylene sorbitan esters (the Tween surfactants),polyoxyethylene ethers, polyoxyethlene esters, etc. One useful splittingprocedure uses the consecutive effects of sodium deoxycholate andformaldehyde, and splitting can take place during initial virionpurification (e.g. in a sucrose density gradient solution). Thus asplitting process can involve clarification of the virion-containingmaterial (to remove non-virion material), concentration of the harvestedvirions (e.g. using an adsorption method, such as CaHPO₄ adsorption),separation of whole virions from non-virion material, splitting ofvirions using a splitting agent in a density gradient centrifugationstep (e.g. using a sucrose gradient that contains a splitting agent suchas sodium deoxycholate), and then filtration (e.g. ultrafiltration) toremove undesired materials. Split virions can usefully be resuspended insodium phosphate-buffered isotonic sodium chloride solution.

Purified influenza virus surface antigen vaccines comprise the surfaceantigens hemagglutinin and, typically, also neuraminidase. Processes forpreparing these proteins in purified form are well known in the art. TheFLUVIRIN™, AGRIPPAL™ and INFLUVAC™ products are influenza subunitvaccines.

Another form of inactivated antigen is the virosome [39] (nucleic acidfree viral-like liposomal particles). Virosomes can be prepared bysolubilization of virus with a detergent followed by removal of thenucleocapsid and reconstitution of the membrane containing the viralglycoproteins. An alternative method for preparing virosomes involvesadding viral membrane glycoproteins to excess amounts of phospholipids,to give liposomes with viral proteins in their membrane.

The invention may also be used to produce live vaccines. Such vaccinesare usually prepared by purifying virions from virion-containing fluids.For example, the fluids may be clarified by centrifugation, andstabilized with buffer (e.g. containing sucrose, potassium phosphate,and monosodium glutamate).

The virus may be attenuated. The virus may be temperature-sensitive. Thevirus may be cold-adapted. These three features are particularly usefulwhen using live virus as an antigen, and reverse genetics isparticularly useful for preparing such strains.

HA is the main immunogen in inactivated influenza vaccines, and vaccinedoses are standardised by reference to HA levels, typically measured bySRID. Existing vaccines typically contain about 15 μg of HA per strain,although lower doses can be used e.g. for children, or in pandemicsituations, or when using an adjuvant. Fractional doses such as ½ (i.e.7.5 μg HA per strain), ¼ and ⅛ have been used, as have higher doses(e.g. 3× or 9× doses [40,41]). Thus vaccines may include between 0.1 and150 μg of HA, per influenza strain, preferably between 0.1 and 50 μge.g. 0.1-20 μg, 0.1-15 μg, 0.1-10 μg, 0.5-5 μg, etc. Particular dosesinclude e.g. about 45, about 30, about 15, about 10, about 7.5, about 5,about 3.75, about 1.9, about 1.5, etc. per strain.

For live vaccines, dosing is measured by median tissue cultureinfectious dose (TCID₅₀) rather than HA content, and a TCID₅₀ of between10⁶ and 10⁸ (preferably between 10^(6.5)-10^(7.5)) per strain istypical.

Influenza strains used with the invention may have a natural HA as foundin a wild-type virus, or a modified HA. For instance, it is known tomodify HA to remove determinants (e.g. hyper-basic regions around theHA1/HA2 cleavage site) that cause a virus to be highly pathogenic inavian species. The use of reverse genetics facilitates suchmodifications.

Influenza virus strains for use in vaccines change from season toseason. In inter-pandemic periods, vaccines typically include twoinfluenza A strains (HIN1 and H3N2) and one influenza B strain, andtrivalent vaccines are typical. The invention may also use pandemicviral strains (i.e. strains to which the vaccine recipient and thegeneral human population are immunologically naïve, in particular ofinfluenza A virus), such as H2, H5, H7 or H9 subtype strains, andinfluenza vaccines for pandemic strains may be monovalent or may bebased on a normal trivalent vaccine supplemented by a pandemic strain.Depending on the season and on the nature of the antigen included in thevaccine, however, the invention may protect against one or more of HAsubtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14,H15 or H16. The invention may protect against one or more of influenza Avirus NA subtypes N1, N2, N3, N4, N5, N6, N7, N8 or N9.

As well as being suitable for immunizing against inter-pandemic strains,the compositions of the invention are particularly useful for immunizingagainst pandemic or potentially-pandemic strains. The characteristics ofan influenza strain that give it the potential to cause a pandemicoutbreak are: (a) it contains a new hemagglutinin compared to thehemagglutinins in currently-circulating human strains, i.e. one that hasnot been evident in the human population for over a decade (e.g. H2), orhas not previously been seen at all in the human population (e.g. H5, H6or 1-19, that have generally been found only in bird populations), suchthat the human population will be immunologically naïve to the strain'shemagglutinin; (b) it is capable of being transmitted horizontally inthe human population; and (c) it is pathogenic to humans. A virus withH5 hemagglutinin type is preferred for immunizing against pandemicinfluenza, such as a H5N1 strain. Other possible strains include H5N3,H9N2, H2N2, H7N1 and H7N7, and any other emerging potentially pandemicstrains. The invention is particularly suitable for protecting againstpotential pandemic virus strains that can or have spread from anon-human animal population to humans, for example a swine-origin H1N1influenza strain. The invention is then suitable for vaccinating humansas well as non-human animals.

Other strains whose antigens can usefully be included in thecompositions are strains which are resistant to antiviral therapy (e.g.resistant to oseltamivir [42] and/or zanamivir), including resistantpandemic strains [43].

Compositions of the invention may include antigen(s) from one or more(e.g. 1, 2, 3, 4 or more) influenza virus strains, including influenza Avirus and/or influenza B virus. Where a vaccine includes more than onestrain of influenza, the different strains are typically grownseparately and are mixed after the viruses have been harvested andantigens have been prepared. Thus a process of the invention may includethe step of mixing antigens from more than one influenza strain. Atrivalent vaccine is typical, including antigens from two influenza Avirus strains and one influenza B virus strain. A tetravalent vaccine isalso useful [44], including antigens from two influenza A virus strainsand two influenza B virus strains, or three influenza A virus strainsand one influenza B virus strain.

Viruses for preparation of influenza vaccines may be grown in eggs or incell culture. The current standard method for influenza virus growth forvaccines uses embryonated SPF hen eggs, with virus being purified fromthe egg contents (allantoic fluid). Cell culture is used to make theOPTAFLU™ product. Cells are preferably cultured in serum-free orprotein-free media for growing virus used for vaccine production.Culturing of cells preferably occurs at a temperature between 30 and 40°C. Cells may be cultured during viral growth at a temperature of between30-36° C. or between 32-34° C. or at 33° C. Incubation of infected cellsin this temperature range results in production of an improved influenzavirus for vaccine use [45]. Virus may be grown on cells in adherentculture or in suspension. Microcarrier cultures can be used. In someembodiments, the cells may thus be adapted for growth in suspension.Growth in cell culture may use the same cell type as was used for thereverse genetics which gave rise to the virus.

A method may include a post-growth step of harvesting and isolation ofviruses or their antigens from culture fluids. During isolation ofviruses or proteins, the cells are separated from the culture medium bystandard methods like separation, filtration or ultrafiltration. Theviruses or their antigens are then concentrated according to methodssufficiently known to those skilled in the art, like gradientcentrifugation, filtration, precipitation, chromatography, etc., andthen purified. It is also preferred according to the invention that theviruses are inactivated during or after purification. Virus inactivationcan occur, for example, by β-propiolactone or formaldehyde at any pointwithin the purification process.

Host Cell DNA

Where virus has been isolated and/or grown on a cell line, it isstandard practice to minimize the amount of residual cell line DNA inthe final vaccine, in order to minimize any oncogenic activity of theDNA. Thus a vaccine composition prepared according to the inventionpreferably contains less than 10 ng (preferably less than 1 ng, and morepreferably less than 100 pg) of residual host cell DNA per dose,although trace amounts of host cell DNA may be present.

It is preferred that the average length of any residual host cell DNA isless than 500 bp e.g. less than 400 bp, less than 300 bp, less than 200bp, less than 100 bp, etc.

Contaminating DNA can be removed during vaccine preparation usingstandard purification procedures e.g. chromatography, etc. Removal ofresidual host cell DNA can be enhanced by nuclease treatment e.g. byusing a DNase. A convenient method for reducing host cell DNAcontamination is disclosed in references 46 & 47, involving a two-steptreatment, first using a DNase (e.g. Benzonase), which may be usedduring viral growth, and then a cationic detergent (e.g. CTAB), whichmay be used during virion disruption. Treatment with an alkylatingagent, such as β-propiolactone, can also be used to remove host cellDNA, and advantageously may also be used to inactivate virions [48].

Pharmaceutical Compositions

Vaccine compositions manufactured according to the invention arepharmaceutically acceptable. They usually include components in additionto the antigens e.g. they typically include one or more pharmaceuticalcarrier(s) and/or excipient(s). As described below, adjuvants may alsobe included. A thorough discussion of such components is available inreference 49.

Vaccine compositions will generally be in aqueous form. However, somevaccines may be in dry form, e.g. in the form of injectable solids ordried or polymerized preparations on a patch.

Vaccine compositions may include preservatives such as thiomersal. It ispreferred, however, that the vaccine should be substantially free from(i.e. less than 5 μg/ml) mercurial material e.g. thiomersal-free[37,50]. Vaccines containing no mercury are more preferred. α-tocopherolsuccinate can be included as an alternative to mercurial compounds [37].Preservative-free vaccines are useful.

To control tonicity, it is preferred to include a physiological salt,such as a sodium salt. Sodium chloride (NaCl) is preferred, which may bepresent at between 1 and 20 mg/ml. Other salts that may be presentinclude potassium chloride, potassium dihydrogen phosphate, disodiumphosphate dehydrate, magnesium chloride, calcium chloride, etc.

Vaccine compositions will generally have an osmolality of between 200mOsm/kg and 400 mOsm/kg, preferably between 240-360 mOsm/kg, and willmore preferably fall within the range of 290-310 mOsm/kg. Osmolality haspreviously been reported not to have an impact on pain caused byvaccination [51], but keeping osmolality in this range is neverthelesspreferred.

Vaccine compositions may include one or more buffers. The pH of avaccine composition will generally be between 5.0 and 8.1, and moretypically between 6.0 and 8.0 e.g. 6.5 and 7.5, or between 7.0 and 7.8.

The vaccine composition is preferably sterile. The vaccine compositionis preferably non-pyrogenic e.g. containing <1 EU (endotoxin unit, astandard measure) per dose, and preferably <0.1 EU per dose. The vaccinecomposition is preferably gluten-free.

Vaccine compositions of the invention may include detergent e.g. apolyoxyethylene sorbitan ester surfactant (known as ‘Tweens’), anoctoxynol (such as octoxynol-9 (Triton X-100) ort-octylphenoxypolyethoxyethanol), a cetyl trimethyl ammonium bromide(‘CTAB’), or sodium deoxycholate, particularly for a split or surfaceantigen vaccine. The detergent may be present only at trace amounts.Thus the vaccine may include less than 1 mg/ml of each of octoxynol-10and polysorbate 80. Other residual components in trace amounts could beantibiotics (e.g. neomycin, kanamycin, polymyxin B).

A vaccine composition may include material for a single immunisation, ormay include material for multiple immunisations (i.e. a ‘multidose’kit). The inclusion of a preservative is preferred in multidosearrangements. As an alternative (or in addition) to including apreservative in multidose compositions, the compositions may becontained in a container having an aseptic adaptor for removal ofmaterial.

Influenza vaccines are typically administered in a dosage volume ofabout 0.5 ml, although a half dose (i.e. about 0.25 ml) may beadministered to children.

Suitable containers for compositions of the invention (or kitcomponents) include vials, syringes (e.g. disposable pre-filledsyringes), nasal sprays, etc. These containers should be sterile. A vialmay include a single dose of vaccine, or it may include more than onedose (a ‘multidose’ vial) e.g. 10 doses. Preferred vials are made ofcolourless glass. A vial may have a cap that permits aseptic removal ofits contents, particularly for multidose vials. Containers may be markedto show a half-dose volume e.g. to facilitate delivery to children. Forinstance, a syringe containing a 0.5 ml dose may have a mark showing a0.25 ml volume.

Where a glass container (e.g. a syringe or a vial) is used, then it ispreferred to use a container made from a borosilicate glass rather thanfrom a soda lime glass.

Where a component is packaged into a syringe, the syringe may have aneedle attached to it. If a needle is not attached, a separate needlemay be supplied with the syringe for assembly and use. Such a needle maybe sheathed. Safety needles are preferred. 1-inch 23-gauge, 1-inch25-gauge and ⅝-inch 25-gauge needles are typical.

Adjuvants

Vaccine compositions of the invention may advantageously include anadjuvant, which can function to enhance the immune responses (humoraland/or cellular) elicited in a subject who receives the composition.Preferred adjuvants comprise oil-in-water emulsions. Various suchadjuvants are known, and they typically include at least one oil and atleast one surfactant, with the oil(s) and surfactant(s) beingbiodegradable (metabolisable) and biocompatible. The oil droplets in theemulsion are generally less than 5 μm in diameter, and ideally have asub-micron diameter, with these small sizes being achieved with amicrofluidiser to provide stable emulsions. Droplets with a size lessthan 220 nm are preferred as they can be subjected to filtersterilization.

Specific oil-in-water emulsion adjuvants useful with the inventioninclude, but are not limited to:

-   -   A submicron emulsion of squalene, Tween 80, and Span 85. The        composition of the emulsion by volume can be about 5% squalene,        about 0.5% polysorbate 80 and about 0.5% Span 85. In weight        terms, these ratios become 4.3% squalene, 0.5% polysorbate 80        and 0.48% Span 85. This adjuvant is known as ‘MF59’ [52-54], as        described in more detail in Chapter 10 of ref. 55 and chapter 12        of ref. 56. The MF59 emulsion advantageously includes citrate        ions e.g. 10 mM sodium citrate buffer.    -   An emulsion of squalene, DL-α-tocopherol, and polysorbate 80        (Tween 80). The emulsion may include phosphate buffered saline.        It may also include Span 85 (e.g. at 1%) and/or lecithin. These        emulsions may have from 2 to 10% squalene, from 2 to 10%        tocopherol and from 0.3 to 3% Tween 80, and the weight ratio of        squalene:tocopherol is preferably ≤1 as this provides a more        stable emulsion. Squalene and Tween 80 may be present volume        ratio of about 5:2 or at a weight ratio of about 11:5. One such        emulsion can be made by dissolving Tween 80 in PBS to give a 2%        solution, then mixing 90 ml of this solution with a mixture of        (5 g of DL-α-tocopherol and 5 ml squalene), then microfluidising        the mixture. The resulting emulsion may have submicron oil        droplets e.g. with an average diameter of between 100 and 250        nm, preferably about 180 nm. The emulsion may also include a        3-de-O-acylated monophosphoryl lipid A (3d-MPL). Another useful        emulsion of this type may comprise, per human dose, 0.5-10 mg        squalene, 0.5-11 mg tocopherol, and 0.1-4 mg polysorbate 80        [57].    -   An emulsion of squalene, a tocopherol, and a Triton detergent        (e.g. Triton X-100). The emulsion may also include a 3d-MPL (see        below). The emulsion may contain a phosphate buffer.    -   An emulsion comprising a polysorbate (e.g. polysorbate 80), a        Triton detergent (e.g. Triton X-100) and a tocopherol (e.g. an        α-tocopherol succinate). The emulsion may include these three        components at a mass ratio of about 75:11:10 (e.g. 750 μg/ml        polysorbate 80, 110 μg/ml Triton X-100 and 100 μg/ml        α-tocopherol succinate), and these concentrations should include        any contribution of these components from antigens. The emulsion        may also include squalene. The emulsion may also include a        3d-MPL (see below). The aqueous phase may contain a phosphate        buffer.    -   An emulsion of squalane, polysorbate 80 and poloxamer 401        (“Pluronic™ L121”). The emulsion can be formulated in phosphate        buffered saline, pH 7.4. This emulsion is a useful delivery        vehicle for muramyl dipeptides, and has been used with        threonyl-MDP in the “SAF-1” adjuvant [58] (0.05-1% Thr-MDP, 5%        squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can        also be used without the Thr-MDP, as in the “AF” adjuvant [59]        (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80).        Microfluidisation is preferred.    -   An emulsion comprising squalene, an aqueous solvent, a        polyoxyethylene alkyl ether hydrophilic nonionic surfactant        (e.g. polyoxyethylene (12) cetostearyl ether) and a hydrophobic        nonionic surfactant (e.g. a sorbitan ester or mannide ester,        such as sorbitan monoleate or ‘Span 80’). The emulsion is        preferably thermoreversible and/or has at least 90% of the oil        droplets (by volume) with a size less than 200 nm [60]. The        emulsion may also include one or more of: alditol; a        cryoprotective agent (e.g. a sugar, such as dodecylmaltoside        and/or sucrose); and/or an alkylpolyglycoside. The emulsion may        include a TLR4 agonist [61]. Such emulsions may be lyophilized.    -   An emulsion of squalene, poloxamer 105 and Abil-Care [62]. The        final concentration (weight) of these components in adjuvanted        vaccines are 5% squalene, 4% poloxamer 105 (pluronic polyol) and        2% Abil-Care 85 (Bis-PEG/PPG-16/16 PEG/PPG-16/16 dimethicone;        caprylic/capric triglyceride).    -   An emulsion having from 0.5-50% of an oil, 0.1-10% of a        phospholipid, and 0.05-5% of a non-ionic surfactant. As        described in reference 63, preferred phospholipid components are        phosphatidylcholine, phosphatidylethanolamine,        phosphatidylserine, phosphatidylinositol, phosphatidylglycerol,        phosphatidic acid, sphingomyelin and cardiolipin. Submicron        droplet sizes are advantageous.    -   An emulsion in which a saponin (e.g. QuilA or QS21) and a sterol        (e.g. a cholesterol) are associated as helical micelles [64].

In some embodiments an emulsion may be mixed with antigenextemporaneously, at the time of delivery, and thus the adjuvant andantigen may be kept separately in a packaged or distributed vaccine,ready for final formulation at the time of use. In other embodiments anemulsion is mixed with antigen during manufacture, and thus thecomposition is packaged in a liquid adjuvanted form.

Methods of Treatment, and Administration of the Vaccine

The invention provides a vaccine manufactured according to theinvention. These vaccine compositions are suitable for administration tohuman or non-human animal subjects, such as pigs, and the inventionprovides a method of raising an immune response in a subject, comprisingthe step of administering a composition of the invention to the subject.The invention also provides a composition of the invention for use as amedicament, and provides the use of a composition of the invention forthe manufacture of a medicament for raising an immune response in asubject.

The immune response raised by these methods and uses will generallyinclude an antibody response, preferably a protective antibody response.

Compositions of the invention can be administered in various ways. Themost preferred immunisation route is by intramuscular injection (e.g.into the arm or leg), but other available routes include subcutaneousinjection or intranasal [65-67].

Vaccines prepared according to the invention may be used to treat bothchildren and adults.

Treatment can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunisation schedule and/or ina booster immunisation schedule. Multiple doses will typically beadministered at least 1 week apart (e.g. about 2 weeks, about 3 weeks,about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12weeks, about 16 weeks, etc.).

Vaccines produced by the invention may be administered to patients atsubstantially the same time as (e.g. during the same medicalconsultation or visit to a healthcare professional or vaccinationcentre) other vaccines.

Similarly, vaccines of the invention may be administered to patients atsubstantially the same time as (e.g. during the same medicalconsultation or visit to a healthcare professional) an antiviralcompound, and in particular an antiviral compound active againstinfluenza virus (e.g. oseltamivir and/or zanamivir).

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x is optional andmeans, for example, x±10%.

Unless specifically stated, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thuscomponents can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined with the third component, etc.

Where animal (and particularly bovine) materials are used in the cultureof cells, they should be obtained from sources that are free fromtransmissible spongiform encephalopathies (TSEs), and in particular freefrom bovine spongiform encephalopathy (BSE). Overall, it is preferred toculture cells in the total absence of animal-derived materials.

MODES FOR CARRYING OUT THE INVENTION

Source influenza viruses are S-OIV strain A/California/4/09 for HA andNA segments and PR/8/34 for the remaining six backbone segments. DNAsequences encoding these eight segments are prepared, with each segmentbeing flanked by a human pol-I promoter at one end and a pol-Iterminator at the other end. These pol-I elements are surrounded by apol-II promoter from CMV and a pol-II terminator sequence and polAsignal. The pol-I promoter drives transcription of a negative senseviral RNA segment with faithful wild-type vRNA termini. The pol-IIpromoter drives transcription of a mRNA encoding the viral protein. DNAsegments for each segment are joined to give a single linear DNAmolecule, ˜24 kbp, encoding the whole reassortant influenza virusgenome. The overall synthesis of this molecule follows the generalmethods disclosed by Gibson et al. in reference 8.

The linear DNA construct is transfected into a culture of MDCK 33016cells. This cell line has been found to recognise the human pol-Ipromoter for influenza virus reverse genetics rescue. Incubation of thetransfected cells soon leads to the appearance of reassortant influenzavirus in the culture medium. This strain (“RG-lin-CA-1”) can be purifiedby conventional methods and used as a seed for vaccine manufacture.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

REFERENCES

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1-16. (canceled)
 17. A reverse genetics system comprising a combinationof at least one plasmid bacterial expression construct and at least onenon-bacterial expression construct, wherein each construct comprisescoding sequences for one or more genome segments of a segmented RNAvirus.
 18. The reverse genetics system of claim 17, wherein the RNAvirus genome segments from the plasmid and the at least onenon-bacterial expression construct are different.
 19. The reversegenetics system of claim 17, wherein the at least one non-bacterialexpression construct is linear.
 20. The reverse genetics system of claim17, wherein the at least one non-bacterial expression construct iscircular.
 21. A eukaryotic host cell comprising the reverse geneticssystem of claim
 17. 22. A bacterial plasmid comprising coding sequencesfor eight different genome segments of an influenza virus, whereinexpression of each segment is controlled by a mammalian pol-I promoteror a bacteriophage polymerase promoter.
 23. A eukaryotic cell comprisingthe bacterial plasmid of claim
 22. 24. A bacterial cell comprising thebacterial plasmid of claim
 22. 25. A method of preparing a eukaryotichost cell, comprising inserting the plasmid and the at least onenon-bacterial expression construct of claim 17 into the eukaryotic hostcell.
 26. A method of preparing a segmented RNA virus, comprisingculturing a host cell under conditions that enable expression ofsegments of the RNA virus from a reverse genetics system comprising acombination of at least one plasmid bacterial expression construct andat least one non-bacterial expression construct, wherein each constructcomprises coding sequences for one or more genome segments of thesegmented RNA virus.
 27. A segmented RNA virus produced by the method ofclaim
 26. 28. A method of preparing a viral vaccine, comprising:infecting a culture host with a segmented RNA virus produced byculturing a host cell under conditions that enable expression ofsegments of the RNA virus from a reverse genetics system comprising acombination of at least one plasmid bacterial expression construct andat least one non-bacterial expression construct, wherein each constructcomprises coding sequences for one or more genome segments of thesegmented RNA virus; growing the segmented RNA virus in the infectedculture host; and preparing a vaccine from the grown virus.
 29. Themethod of claim 28, wherein the culture host is cells.
 30. The method ofclaim 28, wherein the culture host is eggs.
 31. The method of claim 25,wherein the plasmid and the at least one non-bacterial expressionconstruct are inserted into the eukaryotic host cell by any suitabletransfection method.
 32. The eukaryotic cell of claim 23, wherein thebacterial plasmid is inserted into the eukaryotic cell by any suitabletransfection method.
 33. The reverse genetics system of claim 17,wherein the plasmid and the at least one non-bacterial expressionconstruct encode for both viral protein and viral RNA expression. 34.The reverse genetics system of claim 33, wherein the plasmid and the atleast one non-bacterial expression construct each comprise abi-directional expression construct.
 35. The reverse genetics system ofclaim 17, wherein the segmented RNA virus is an influenza virus.
 36. Themethod of claim 26, wherein the segmented RNA virus is an influenzavirus.
 37. The method of claim 28, wherein the segmented RNA virus is aninfluenza virus.